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==Characteristics==
==Characteristics==
''Serratia'' nuclease was first purified from its native source in 1969.<ref name="Nestle1969">{{cite journal | last=Nestle | first=Marion | last2=Roberts | first2=W.K. | title=An Extracellular Nuclease from Serratia marcescens | journal=Journal of Biological Chemistry | publisher=Elsevier BV | volume=244 | issue=19 | year=1969 | issn=0021-9258 | doi=10.1016/s0021-9258(18)63648-8 | pages=5213–5218}}</ref> It was cloned in 1987 and shown to consist of a 266 [[Protein precursor|protein precursor]],<ref name="Ball Saurugger Benedik 1987 pp. 183–192">{{cite journal | vauthors = Ball TK, Saurugger PN, Benedik MJ | title = The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli | journal = Gene | volume = 57 | issue = 2–3 | pages = 183–192 | year = 1987 | pmid = 3319779 | doi = 10.1016/0378-1119(87)90121-1 | publisher = Elsevier BV }}</ref> which is further cleaved and secreted as a 245 amino acid active nuclease.<ref name="Biedermann1989">{{cite journal | vauthors = Biedermann K, Jepsen PK, Riise E, Svendsen I | title = Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli | journal = Carlsberg Research Communications | volume = 54 | issue = 1 | pages = 17–27 | year = 1989 | pmid = 2665765 | doi = 10.1007/bf02910469 | publisher = Springer Science and Business Media LLC | s2cid = 12831178 }}</ref> It has two disulfide bonds, the first between cysteine 30 & 34 and the second between cysteine 222 & 264<ref name="Biedermann1989"></ref>. Reduction of these disulfides or site directed mutagenesis of their residues to serine, specifically the first one, leads to a large loss in nuclease activity,<ref name="Benedik1998">{{cite journal | vauthors = Benedik MJ, Strych U | title = Serratia marcescens and its extracellular nuclease | journal = FEMS Microbiology Letters | volume = 165 | issue = 1 | pages = 1–13 | date = August 1998 | pmid = 9711834 | doi = 10.1111/j.1574-6968.1998.tb13120.x | publisher = Oxford University Press (OUP) }}</ref> and a loss of the ability to reversibly regain activity after inactivating 40-60˚C heat treatments.<ref name="Biedermann1989">{{cite journal | vauthors = Biedermann K, Jepsen PK, Riise E, Svendsen I | title = Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli | journal = Carlsberg Research Communications | volume = 54 | issue = 1 | pages = 17–27 | year = 1989 | pmid = 2665765 | doi = 10.1007/bf02910469 | publisher = Springer Science and Business Media LLC | s2cid = 12831178 }}</ref>. It has a much higher catalytic efficiency than other nucleases, about 4 times greater than [[Micrococcal nuclease|staphylococcal nuclease]], and about 34 times greater than [[Deoxyribonuclease I|bovine pancreatic DNase I]].<ref name="Benedik1998"></ref> The enzyme cleaves single or double stranded DNA and RNA with similar rates, so long as the substrate DNA or RNA contains no fewer than 5 nucleotides (or basepairs).<ref name="Benedik1998"></ref> [[Magnesium|Magnesium (II)]] (Mg<sup>2+</sup>) is an essential cofactor for its nuclease activity.<ref name="Benedik1998"></ref> ''Serratia'' nuclease is activated by up to 4M [[urea]].<ref name="EMD datasheet>{{cite web |title=Benzonase® Nuclease - Effective removal of nucleic acids and viscosity reduction from protein solutions |url= https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/342/154/benzonase-nuclease-ms.pdf | work = EMD Biosciences | publisher = SigmaAldrich |access-date=29 April 2023}}</ref> At 5M urea the initial activity is decreased from its peak although still above its baseline, and the enzyme is significantly inhibited after 60 minutes. At 6M urea, the nuclease activity is below baseline and almost completely inactivated within 60 minutes. At 7M the nuclease becomes essentially completely inactivated within 15 minutes, but significant and workable degradation of nucleic acids can occur before the nuclease is inactivated.<ref name="EMD datasheet></ref> 8M urea causes a complete inactivation of the enzyme within 5 minutes.<ref name="Biedermann1989"></ref>
''Serratia'' nuclease was first purified from its native source in 1969.<ref name="Nestle1969">{{cite journal | last=Nestle | first=Marion | last2=Roberts | first2=W.K. | title=An Extracellular Nuclease from Serratia marcescens | journal=Journal of Biological Chemistry | publisher=Elsevier BV | volume=244 | issue=19 | year=1969 | issn=0021-9258 | doi=10.1016/s0021-9258(18)63648-8 | pages=5213–5218}}</ref> It was cloned in 1987 and shown to consist of a 266 [[Protein precursor|protein precursor]],<ref name="Ball Saurugger Benedik 1987 pp. 183–192">{{cite journal | vauthors = Ball TK, Saurugger PN, Benedik MJ | title = The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli | journal = Gene | volume = 57 | issue = 2–3 | pages = 183–192 | year = 1987 | pmid = 3319779 | doi = 10.1016/0378-1119(87)90121-1 | publisher = Elsevier BV }}</ref> which is further cleaved and secreted as a 245 amino acid active nuclease.<ref name="Biedermann1989">{{cite journal | vauthors = Biedermann K, Jepsen PK, Riise E, Svendsen I | title = Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli | journal = Carlsberg Research Communications | volume = 54 | issue = 1 | pages = 17–27 | year = 1989 | pmid = 2665765 | doi = 10.1007/bf02910469 | publisher = Springer Science and Business Media LLC | s2cid = 12831178 }}</ref> Its active form in solution is a homodimer.<ref name="Benedik1998">{{cite journal | vauthors = Benedik MJ, Strych U | title = Serratia marcescens and its extracellular nuclease | journal = FEMS Microbiology Letters | volume = 165 | issue = 1 | pages = 1–13 | date = August 1998 | pmid = 9711834 | doi = 10.1111/j.1574-6968.1998.tb13120.x | publisher = Oxford University Press (OUP) }}</ref> It has two disulfide bonds, the first between cysteine 30 & 34 and the second between cysteine 222 & 264.<ref name="Biedermann1989"></ref>. Reduction of these disulfides or site directed mutagenesis of their residues to serine, specifically the first one, leads to a large loss in nuclease activity,<ref name="Benedik1998"></ref> and a loss of the ability to reversibly regain activity after inactivating 40-60˚C heat treatments.<ref name="Biedermann1989">{{cite journal | vauthors = Biedermann K, Jepsen PK, Riise E, Svendsen I | title = Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli | journal = Carlsberg Research Communications | volume = 54 | issue = 1 | pages = 17–27 | year = 1989 | pmid = 2665765 | doi = 10.1007/bf02910469 | publisher = Springer Science and Business Media LLC | s2cid = 12831178 }}</ref> It has a much higher catalytic efficiency than other nucleases, about 4 times greater than [[Micrococcal nuclease|staphylococcal nuclease]], and about 34 times greater than [[Deoxyribonuclease I|bovine pancreatic DNase I]].<ref name="Benedik1998"></ref> The enzyme cleaves single or double stranded DNA and RNA with similar rates, so long as the substrate DNA or RNA contains no fewer than 5 nucleotides (or basepairs).<ref name="Benedik1998"></ref> [[Magnesium|Magnesium (II)]] (Mg<sup>2+</sup>) is an essential cofactor for its nuclease activity.<ref name="Benedik1998"></ref> ''Serratia'' nuclease is activated by up to 4M [[urea]].<ref name="EMD datasheet>{{cite web |title=Benzonase® Nuclease - Effective removal of nucleic acids and viscosity reduction from protein solutions |url= https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/342/154/benzonase-nuclease-ms.pdf | work = EMD Biosciences | publisher = SigmaAldrich |access-date=29 April 2023}}</ref> At 5M urea the initial activity is decreased from its peak although still above its baseline, and the enzyme is significantly inhibited after 60 minutes. At 6M urea, the nuclease activity is below baseline and almost completely inactivated within 60 minutes. At 7M the nuclease becomes essentially completely inactivated within 15 minutes, but significant and workable degradation of nucleic acids can occur before the nuclease is inactivated.<ref name="EMD datasheet></ref> 8M urea causes a complete inactivation of the enzyme within 5 minutes.<ref name="Biedermann1989"></ref>


===Optimal conditions<ref name="EMD datasheet></ref>===
===Optimal conditions<ref name="EMD datasheet></ref>===

Revision as of 19:01, 30 April 2023

Serratia marcescens nuclease
Identifiers
EC no.3.1.30.2
CAS no.9025-65-4
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Serratia marcescens nuclease
Identifiers
OrganismSerratia marcescens
SymbolnucA
UniProtP13717
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Serratia marcescens nuclease (EC 3.1.30.2, endonuclease (Serratia marcescens), barley nuclease, plant nuclease I, nucleate endonuclease) is an enzyme.[1][2][3][4] This enzyme catalyses the following chemical reaction

Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end-products

Hydrolyses double- or single-stranded substrate DNA or RNA. It is a representative of the DNA/RNA non-specific endonuclease family.

It is commercially available.

Characteristics

Serratia nuclease was first purified from its native source in 1969.[5] It was cloned in 1987 and shown to consist of a 266 protein precursor,[6] which is further cleaved and secreted as a 245 amino acid active nuclease.[7] Its active form in solution is a homodimer.[8] It has two disulfide bonds, the first between cysteine 30 & 34 and the second between cysteine 222 & 264.[7]. Reduction of these disulfides or site directed mutagenesis of their residues to serine, specifically the first one, leads to a large loss in nuclease activity,[8] and a loss of the ability to reversibly regain activity after inactivating 40-60˚C heat treatments.[7] It has a much higher catalytic efficiency than other nucleases, about 4 times greater than staphylococcal nuclease, and about 34 times greater than bovine pancreatic DNase I.[8] The enzyme cleaves single or double stranded DNA and RNA with similar rates, so long as the substrate DNA or RNA contains no fewer than 5 nucleotides (or basepairs).[8] Magnesium (II) (Mg2+) is an essential cofactor for its nuclease activity.[8] Serratia nuclease is activated by up to 4M urea.[9] At 5M urea the initial activity is decreased from its peak although still above its baseline, and the enzyme is significantly inhibited after 60 minutes. At 6M urea, the nuclease activity is below baseline and almost completely inactivated within 60 minutes. At 7M the nuclease becomes essentially completely inactivated within 15 minutes, but significant and workable degradation of nucleic acids can occur before the nuclease is inactivated.[9] 8M urea causes a complete inactivation of the enzyme within 5 minutes.[7]

Optimal conditions[9]

Condition Optimal1 Effective2
Mg2+ concentration 1 - 2 mM 1 - 10 mM
pH 8.2 - 9.2 6.0 - 10.0
Temperature 37˚C 0 - 42˚C
Dithiothreitol (DTT) < 100 mM > 100 mM
β-Mercaptoethanol (BME) < 100 mM > 100 mM
Monovalent cation concentration (Na+, K+, etc.) 0 - 20 mM 0 - 150 mM
PO43- 0 - 10 mM 0 - 100 mM
Urea < 4M > 4M

1="Optimal" is the condition in which Serratia nuclease retains >90 % of its activity.

2="Effective" is the condition in which Serratia nuclease retains >15 % of its activity.

Inhibitory conditions

Some inhibitory conditions are known:[9]

Use in biotechnology

Given its high activity, high stability & reversible inactivation to heat treatments, rate enhancement or otherwise compatibility with some denaturing reagents like urea, Serratia nuclease was recognized early on to have industrial & commercialization potential. A patent covering the recombinant expression of Serratia nuclease in E. coli was submitted by Benzon Pharma in 1986, granted in 1992, & expired in 2006.[10] This recombinant Serratia nuclease was commercialized as Benzonase, and is still available from and a registered trademark of Merck KGaA.[11] Notably, the patented sequence[10][12] for Benzonase is slightly different (1 amino acid substitution) from the Serratia marcescens nuclease which was cloned publicly.[13]

As the benzonase patent is now expired, and in fact was never submitted nor granted in the United States, several commercial alternatives for recombinantly produced Serratia marcescens nuclease are now available:

(A current notable non-producer is New England Biolabs)[23]

See also

References

  1. ^ Mikulski AJ, Laskowski M (October 1970). "Mung bean nuclease I. 3. Purification procedure and (3') omega monophosphatase activity". The Journal of Biological Chemistry. 245 (19): 5026–5031. doi:10.1016/S0021-9258(18)62813-3. PMID 4319109.
  2. ^ Stevens A, Hilmoe RJ (1960). "Studies on a nuclease from Azotobacter agilis. I. Isolation and mode of action". Journal of Biological Chemistry. 235 (10): 3016–3022. doi:10.1016/S0021-9258(18)64581-8.
  3. ^ Stevens A, Hilmoe RJ (1960). "Studies on a nuclease from Azotobacter agilis. II. Hydrolysis of ribonucleic and deoxyribonucleic acids". Journal of Biological Chemistry. 235 (10): 3023–3027. doi:10.1016/S0021-9258(18)64582-X.
  4. ^ Wechter WJ, Mikulski AJ, Laskowski M (February 1968). "Gradation of specificity with regard to sugar among nucleases". Biochemical and Biophysical Research Communications. 30 (3): 318–322. doi:10.1016/0006-291x(68)90453-1. PMID 4296679.
  5. ^ Nestle, Marion; Roberts, W.K. (1969). "An Extracellular Nuclease from Serratia marcescens". Journal of Biological Chemistry. 244 (19). Elsevier BV: 5213–5218. doi:10.1016/s0021-9258(18)63648-8. ISSN 0021-9258.
  6. ^ Ball TK, Saurugger PN, Benedik MJ (1987). "The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli". Gene. 57 (2–3). Elsevier BV: 183–192. doi:10.1016/0378-1119(87)90121-1. PMID 3319779.
  7. ^ a b c d Biedermann K, Jepsen PK, Riise E, Svendsen I (1989). "Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli". Carlsberg Research Communications. 54 (1). Springer Science and Business Media LLC: 17–27. doi:10.1007/bf02910469. PMID 2665765. S2CID 12831178.
  8. ^ a b c d e Benedik MJ, Strych U (August 1998). "Serratia marcescens and its extracellular nuclease". FEMS Microbiology Letters. 165 (1). Oxford University Press (OUP): 1–13. doi:10.1111/j.1574-6968.1998.tb13120.x. PMID 9711834.
  9. ^ a b c d "Benzonase® Nuclease - Effective removal of nucleic acids and viscosity reduction from protein solutions" (PDF). EMD Biosciences. SigmaAldrich. Retrieved 29 April 2023.
  10. ^ a b EP 0229866A1, Molin S, Givskov M, Riise E, "Bacterial enzymes and method for their productio", issued 9 December 1992, assigned to Benzon Pharma AS and Takeda Pharma AS 
  11. ^ "Benzonase® Nuclease HC, Purity > 99% - 71206". MilliporeSigma. Retrieved 2023-04-29.
  12. ^ "UniProt". UniProt. Retrieved 2023-04-29.
  13. ^ "UniProt". UniProt. Retrieved 2023-04-29.
  14. ^ "Basemuncher Benzonase". Westburg. Retrieved 2023-04-29.
  15. ^ "Benzo Nuclease". Tinzyme Ltd – Enzymes, dNTP and rNTP. 2021-12-24. Retrieved 2023-04-29.
  16. ^ "Benz-Neburase™, His". GenScript. 2021-08-12. Retrieved 2023-04-29.
  17. ^ "B-1400-5KU - Decontaminase™, 5 KU". AG Scientific. 2022-12-13. Retrieved 2023-04-29.
  18. ^ "Denarase". c-LEcta. 2022-12-13. Retrieved 2023-04-29.
  19. ^ "Benzonase Nuclease Alternative, DENARASE Nuclease Alternative". Syd Labs. 2020-05-01. Retrieved 2023-04-29.
  20. ^ "GENIUS™Nuclease DMF Filed". ACROBiosystems. Retrieved 2023-04-29.
  21. ^ "Pierce™ Universal Nuclease for Cell Lysis". Thermo Fisher Scientific. 2023-04-29. Retrieved 2023-04-29.
  22. ^ "TurboNuclease". Accelagen. 2023-04-29. Retrieved 2023-04-29.
  23. ^ Biolabs, New England. "DNA Modifying Enzymes & Cloning Technologies - Exonucleases and Non-specific Endonucleases". New England Biolabs. Retrieved 30 April 2023.
allikas: https://en.wikipedia.org/wiki/Special%3ADiff%2F1152521346